Related Experiment Videos
Energy transfer analysis of Fos-Jun dimerization and DNA binding
L R Patel1, T Curran, T K Kerppola
1Roche Institute of Molecular Biology, Nutley, NJ 07110.
Summary
Proto-oncogenes Fos and Jun form rapid dimers and bind DNA quickly. While free Fos-Jun subunits exchange readily, DNA binding stabilizes the complex, preventing subunit exchange.
Area of Science:
- Molecular Biology
- Protein Interactions
- Gene Regulation
Background:
- Proto-oncogenes Fos and Jun are transcription factors that regulate gene expression.
- Their function relies on forming dimeric complexes mediated by a leucine zipper.
- These dimers possess a DNA-binding domain crucial for their activity.
Purpose of the Study:
- To quantitatively analyze the dimerization and DNA binding of Fos and Jun proteins in solution.
- To investigate the kinetics of Fos-Jun complex formation and subunit exchange.
- To understand how DNA binding affects the stability of Fos-Jun dimers.
Main Methods:
- Developed a resonance energy transfer (RET) assay for quantitative analysis.
- Labeled Fos and Jun polypeptides with fluorescein and rhodamine fluorophores on cysteine residues.
- Measured RET efficiency changes upon protein dimerization and DNA binding.
Main Results:
- RET assay demonstrated efficient heterodimer formation and DNA binding.
- DNA binding induced a conformational change, reducing fluorophore distance by ~3 Å.
- Fos-Jun dimerization and DNA binding kinetics were rapid (half-times < 10 s).
- Subunit exchange was rapid in free solution but significantly inhibited upon DNA binding.
Conclusions:
- Fos and Jun dimerization and DNA binding are rapid processes.
- Leucine zipper transcription factors exhibit dynamic subunit exchange in solution.
- DNA binding confers significant stability to the Fos-Jun complex, inhibiting subunit exchange.